Division of Legal Medicine, Department of Social Medicine, Nihon University School of Medicine, Tokyo, Japan.
Electrophoresis. 2010 Jul;31(14):2411-5. doi: 10.1002/elps.200900754.
We developed a direct and rapid method for the diagnosis of death by drowning by PCR amplification of phytoplankton DNA using human tissues. The primers were designed based on the DNA sequence of the 16S ribosomal RNA gene (16S rDNA) of Cyanobacterium. Samples of lung, liver and kidney tissues were collected from 53 autopsied individuals diagnosed as death by drowning. Without DNA extraction, the tissue fragments were incubated directly in a digest buffer developed in this study, for 20 min. Using 1 microL of the tissue digest solution in PCR, the 16S rDNA was successfully amplified. The specific 16S rDNA fragment was identified from the standard picoplankton Euglena gracilis, the tissues of bodies died from drowning and water samples from the drowning scenes. On the other hand, no PCR products were found in the tissues of individuals who died from causes other than drowning. Various quantities of tissue weighing 1, 5, 10, 20 and 30 mg were tested, and the PCR amplification detected the specific 16S rDNA fragment from all the quantities of tissue tested. This method was found to be more reliable, sensitive, specific and rapid when compared to the conventional diagnosis of death by drowning using the diatom test by acid digestion method.
我们开发了一种直接、快速的方法，通过聚合酶链反应（PCR）扩增人类组织中的浮游植物 DNA 来诊断溺亡。引物是根据蓝藻的 16S 核糖体 RNA 基因（16S rDNA）的 DNA 序列设计的。从 53 例经尸检诊断为溺亡的个体中采集了肺、肝和肾组织样本。无需提取 DNA，组织片段直接在本研究中开发的消化缓冲液中孵育 20 分钟。使用 1μL 组织消化液进行 PCR，成功扩增了 16S rDNA。从标准微微型浮游生物绿眼虫、溺亡者的组织和溺亡现场的水样中鉴定出了特定的 16S rDNA 片段。另一方面，在非溺亡原因导致死亡的个体组织中未发现 PCR 产物。我们测试了重量为 1、5、10、20 和 30mg 的各种组织量，PCR 扩增从所有测试组织量中检测到了特定的 16S rDNA 片段。与传统的酸消化法硅藻试验相比，这种方法在诊断溺亡方面更可靠、更敏感、更特异、更快。
